JPH03500788A - Oligosaccharide heparin fragments as inhibitors of the complement cascade - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] as an inhibitor of the complement cascade Oligosaccharide heparin fragment Five minutes ■ This invention is a continuation-in-part of US Patent Application No. 717.213.

The present invention inhibits the complement cascade without excessive side effects of anticoagulant activity. Compounds and pharmaceutical compositions. This compound contains about 6 saccharide units ~ A smaller one of the larger polysaccharide heparin, about 16 sunkalide units. It is a chain fragment.

Natsu Yikei Heparin is a highly sulfated polydisperse α-(1→ 4) - is a bonded copolymer. Jaques.

It is synthesized as a proteoglycan with a molecular weight of . Heparin binds to protein core do. This protein core consists of the gelcosaminoglycan heparin (average molecular weight 10.0 00 to 14,000 Daltons).

Heparin's primary use is as an anticoagulant. However, heparin It has many biological activities, including (1)° angiogenesis; (Folkman et al., 5science 221) (2) ability to control other cell proliferation and growth processes , (3) the ability to activate and release plasma riboprotein lipase (Merc Hant et al., 匡读dan卩婬婬: 151-58.1984), and (4) Suppl. including the ability to inhibit the body cascade.

In 1929, Ecker and Gross first identified heparin, which controls complement activation. demonstrated his ability to Next, other researchers discovered that heparin can be used to and other amplification pathways. The anticoagulant activity of heparin is antithrombin ■Specific oligosaccharide sequences on heparin polymers that can bind to mediated by. However, the structure-activity of heparin on the complement cascade system The relationship is still poorly understood.

The complement cascade inhibitory activity of heparin is used in various ways to reduce host immune responses. Can be used for functions.

Organ transplant rejection is a corresponding immune response that is unfavorable to the individual host (i.e. mammalian). It is. Similarly, various types of autoimmunity arise from an immune response that attacks an individual's own cells. A disease exists. These immune and autoimmune diseases cannot be effectively treated. Represents a group of often difficult diseases. There are many drugs that can be immunosuppressants. However, suitable agents for modulating or specifically inhibiting complement cascade activation There are currently no drugs available. Complement inhibition is an important player in various immunodeficiencies. It's a point of view. Diseases in which substances that inhibit the complement cascade system are therapeutically useful sudden nocturnal haematuria, rheumatoid arthritis (in this case, avoidance of complement activation) for which the substance would be administered directly into the joint sac), and hereditary angioedema (angioedma) (in this case, lack of complement regulatory proteins leads to active complement consumption ) is included. Other diseases include exsanguination, rheumatoid arthritis, and systemic erythema. Includes patchy lupus.

The anticoagulant activity of heparin is due to the special properties in its structure for the binding of antithrombin. It has been shown to be associated with the presence of heterogeneous oligosaccharide sequences.

After binding to heparin, antithrombin is able to inhibit numerous blood clotting factors. and therefore prevent blood clotting.

Heparin's anticomplement activity is mediated by binding to various complement cascade proteins; and thereby the classical route and the alternative (a1 tern) ate) Control both routes. Heparin and heparin oligosaccharide , cell-bound amplification pathway C3 convertases, C3b, Inhibits the production of Bb, C3b, Bb, P and C3b, Bb, Nef. By inhibiting part of the complement cascade. The anticoagulant activity of heparin is on C3b. interfering with the binding site of Furthermore, heparin is a fluid of B due to D in the presence of C3b. It prevents phase consumption and also shows a direct effect on C3b.

Therefore, it has heparin-like anticomplement activity, and the anticoagulant activity of heparin on a weight basis. There is a need in the art for therapeutic agents with significantly reduced (e.g., 10% or less) exist. The present invention fills this need.

home road The abbreviations for complement used herein are C3 for the third complement protein; C3b for the activated third complement protein, and C3b for the activated fourth complement protein. In most cases, C4b is used, and B, P and the letter component (le) of the alternative amplification pathway of complement are used. ter component). Other abbreviations include surface C4b and C3b EAC4b, 3b for ovine erythroid cell intermediates containing. E AC4b, 3b', EAC4b, 3b", and EAC4b, 3b"'c, 1 ×109 EAcl, 5■ per 4b, 20I! EA using g and 1100 IJ CI, refers to the cellular intermediate produced from 4b. About Satsuka Ride , ΔUA is 4-deoxy-α-L-threo-hex-4-enopyronosyluro p means pyranose, and GlcA means glucuronic acid (non-reducing sugar). , IdoA means iduronic acid, And S means sulfate.

Main skin ■M-jo In summary, the present invention provides compounds having a degree of polymerization of from about 6 to about 24 saccharide units. Regarding the palin oligosaccharide fraction.

The oligosaccharide of the present invention can have an even number of saccharide units. , where the group of saccharide units is disaccharide, tetrasaccharide and hexasaccharide.

The present invention also provides for oligomers having an odd degree of polymerization from about 5 to about 23 saccharide units. Regarding the gosatucharide fraction, here the non-reducing end (Δ[!Ap) sactucaride has been removed. Removal of non-reducing terminal saccharides is performed using an even number of oligosaccharides. by treatment with ozone at acidic pH, as described herein. can be achieved.

Preferably, the disancalide group of the saccharide unit is a trisulfate. Todisaccharide, for example ΔUAp25 (1 → 4) -a: -D-GIcN p2S6S (1 in Table 1 and when n=0 in formula I). sankara The tetrasaccharide group of the id unit is pentasulfate tetrasaccharide. Kalide, for example ΔUAp2S(1→4)-α-D-GlcNp2S(1→ 4) -a -L -IdoAp2S -a -D-GlcNp2S6S (No. Table 1 middle native) and ΔUAp2S (1 = 4) -cr -D -GlcNp2 S6S(1→4)-β-D-GlcAp(1→4)-cx-D-G1c Np2S6S (1 in Table 1), and hexasulfate tetrasaccharide , for example ΔUAp2S(1→4)-α-D-GlcNp2S6S(1→4)- α-L-IdoAp2S(1-+4)-a-D-GlcNp2S6S( selected from the group consisting of The saccharide group includes septasulfate hexasaccharides, such as ΔUA p2S(1→4)-cx-D-GlcNp2S6S(1-+4)-α-L -1doAp(1→4) o knee D -GIcNAcp6S-(1-)4)β- D - GlcAp (1 → 4) -o: -D-G1cNp2S3S6S (first -19 in the table.

An example of a trisulfate disaccharide is α-D-2-deoxy-2-amino- 4-deoxy-α linked 1→4 to glucopyranosyl-2,6-disulfate -L-threo-hex-4-enopyronosyluronic acid-2-sulfate , where α and β indicate the anomeric configuration of the sugar, and D and L indicates the absolute configuration of the sugar.

Most preferably hexasaccharides, octasaccalides and decasacucalides is a trisulfate disaccharide, heparin disancharide (2). Decasa having tri-, tetra- and penta-oligomers, e.g. structure (I) Tsukaride IOD (Table 1).

The method of the present invention is suitable for use in polymers having an even degree of polymerization of from about 6 to about 24 saccharide units. Satucharide uni polymerized (polymerized) to form Rigosaccharide A group of cuts were prepared. The method of the invention further provides about 5 to about 23 saccharides. Step of terminal non-reducing sugar removal to form saccharides with odd degree of polymerization of units including. Oligosaccharides are highly sulfated. For example, Natori The % of sulfur is calculated in terms of molecular weight (%) using um salt. has more than 14% sulfur.

The oligosaccharide of the present invention has strong anti-complement activity comparable to that of heparin on a molecular basis. has. The oligosaccharide of the present invention also has anticoagulant activity of heparin on a weight basis. exhibits decreased anticoagulant activity, defined as less than 10% of the

Plates■Plates Figure 1 shows the depolymerization of heparin catalyzed by heparinase to 30% completion. Strong ion exchange of heparin-oligosaccharide mixture prepared by -H Shows PLC. From the column? Iwata Heparin-oligosaccharide sample ( The absorbance at 232 nm of A) and 4 (B) is plotted against the elution time (min). It was cut. Label the peaks collected from the column to identify the sample. (sample number corresponds to degree of polymerization).

Figure 2 shows heparin for the alternative amplification pathway of complement. and the effects of structurally characterized heparin-oligosaccharides. quantity The response curve is heparin (○), septasulfate hexasaccharide 6A (+) , hexasulfate tetrasancalide-4C(mu), pentasulfate Trisaccharide-4B (oral), and trisulfate disaccharide-4B (- ) is indicated.

Figure 3 shows the lysis of EAC4b,3b'' by heparin-oligosaccharide. Shows inhibition. The activity is ((ED50 heparin/ED5Q heparin-oligosatsuka) Ride)X100). ED50 is the average molecular weight of heparin: 14 ,000 on a molecular basis.

Figure 4 shows 41! g heparin and homogeneous structurally defined heparin-oligos Amplification path C3 converter for EAC4b, 3b”° by Sasikaride Figure 2 shows inhibition of convertase production. Heparin Oligosatsuka To the ride day, l, 6C. 8 people.

Dandan, 8C, 8D. Go to 1st stand. IOB, IOC and river are listed in Table 1 of this specification. Defined.

Figure 5 shows various commercially available heparins and heparin oligosaccharides (listed in Table 1). Inhibition of dissolution of EAC4b, 3b” by (shown) on anticoagulant activity Show what is plotted. Anticoagulant activity is standardized on a weight basis as shown in Table 2. Anti-Factor-IIa Amytolysis Assay Compared to Target Porcine Heparin (Hl) It was measured by Inhibition of complement activity is on a weight basis ((ED50 heparin/ED5 0 heparin-oligosaccharides) x 100). Butahepa phosphorus (■H1→H6), bovine heparin (H7, H8), low molecular weight heparin (・ H9), and heparin-oligosaccharide (◆2-+16　b　) are adjacent Identified by number.

Yu Kaifu annotation Partial enzymatic depolymerization of heparin follows the formula: where R is a metal cation or for nonmetallic cations or free acids, X is H or SOJ; Y is HlCOCI-b alkyl or 5o3R, and n is from about 1 to about 11 is an integer) Controlled and reproducible distribution of heparin-oligosaccharides expressed as brought about. Preferably Y is C0CH:l, n is 4 and R is Hydrogen, sodium or potassium.

It has a degree of polymerization of 6 units to 24 units and has a polymerization degree of 2.4 or 6 units. The heparin oligosancalide formed by polymerizing the It has complement inhibitory activity comparable to native heparin on a weight basis and anticoagulant activity. It was found that there was little or no

At 30% reaction completion, the heparin-oligosaccharide mixture was Maximum concentration of heparin-oligosancalide with degree of polymerization of tucharide unit , where all heparin-oligosancharides are even-numbered satukaras. It contained an id unit. This oligosaccharide mixture is a strong anion exchanger. - a defined degree of polymerization fractionated on the basis of charge using HPLC (as shown in the figure) purified heparin-oligosancalide sample containing a single major component can be obtained. ) The elution order from the IPLC column depends on the degree of polymerization, i.e. Disaccharide, followed by tetrasaccharide, followed by hexasaccharide ), and within a size group the elution order depends on the degree of sulfation (i.e. Tetrasaccharides 4A and 4B in Table 1 having 5 sulfates.

Next is the tetrasaccharide 4C with 6 sulfates). Oligosaccharide formation The fraction was re-fractionated to produce heparin-oligosancalide of sufficient purity and A compositional analysis shown in Table 1 was obtained.

“Male Worker” – Ippon Structure of heparin oligosaccharide with a degree of polymerization of 6 to 10 Oligosan force Sulfate gosan force Ride Ride composition (1+ b) Molecular weight Number of bases 6D” 2. Soil C19959 8A 2, 2.4A 2456 11 Publisher: Lord, Shadow! , Dojin 3223 14th edition I, Kusa, Kusa, Dotan 3223 14th issue 2, 2, 6 C316313 (a) The sequence is the basic oligosatellite does not imply the order of the codes.

(b) The structure of the basic oligosaccharides that make up each temporary oligosaccharide is It is as follows.

I Nitrisulfate disaccharide ΔUAp2S (1 → 4) -α-D - GlcNp2S6S; Friend: Pentasulfate tetrasaccharide ΔUAp 2S (1 → 4) -cx-D-G1cNp2S (1 → 4) tx -D -GI cNp2S6S; Saturday: Pentasulfate tetrasaccharide ΔUAp2S (1=4) a knee D-C1cNp2S6S(1→4)-β-D-GlcAp(1→4)-α-D -GlcNp2S6S, ;4C: Hexasulfate tetrasaccharide Δ UAp2S (1 → 4) -tx-D-GIcNp2S6S (1 → 4) (X-L -1doAp2S (1 → 4) cx-D-GlcNp2S6S; Dandan: Septasa Rufeto hexasaccharide ΔUAp2S (1 → 4) -cx-D -C1 cNp2S6S(1→4)-αL-1do8p(1→4)-α-D-G1 cNAcp6S-(1→4)-β-D-G1cAp(1→4)-cx-D -G1cNp2S3S6S.

(C) The oligosaccharide composition of this heparin-oligosaccharide is one form. It can only be placed in This is because 4C=2.2 and this oligosaccharide This is because the sequence of is established.

Hexasacucalide 6D, octasacucalide 8D, and decasacucalide IO Only one arrangement for D is possible. Because their composition analysis shows Apparently these are simply oligomers of heparin disaccharide 2. Ru.

The compounds of the invention inhibit complement activation and have anticoagulant activity, as shown in Table 2. characterized by the ability to have little or no

Oligosaccharides having a degree of polymerization of about 2 to 24 oligosaccharide units are can be produced by partial depolymerization of heparin using heparinase. Ru. Varying the degree of depolymerization (i.e., % reaction completion) from about 1% to about 100%. to optimize the production of oligosaccharides with a specific size or degree of polymerization. Can be done. After partial or complete depolymerization, the oligosaccharide mixture is exposed to strong shade. Ion exchange HPLC column or other suitable technique, e.g. polyacrylamide Fractionation using gel electrophoresis (Rice et al., Biochem, J., 244 :515-22 (1987)]. One or a combination of these techniques Refractionation by fractionation yields highly purified oligosaccharides. 6 Satsukara Oligosaccharides larger than the id unit (i.e., larger than 1000 Daltons) large molecular weight) is sufficient for deionized water using controlled pore dialysis membranes. Desalted by dialysis.

Mejishi table Anticoagulant activity (a) of hevarin-oligosancalide is 167 units/mg Based on a standard curve using heparin (average molecular weight 14,000) with an activity of Determined by

(b)nd, not determined.

The compounds of the invention are obtained by depolymerization of heparin by the lytic enzyme heparinase. . The mixture of oligosaccharides obtained by heparinase digestion is on a weight basis or Desired anti-complement activity on a molecular basis and significantly reduced compared to native heparin or have no anticoagulant activity. Heparinase digestion and The oligosaccharide produced by the subsequent purification contains about 6 to about 24 saccharides. knit and contains an even number of saccharide units. Preferably, ori Gosaccharides have 16-24 saccharide units. Most preferably, Oligosaccharide is an oligosaccharide consisting of 24 saccharides. .

Compounds of the invention also include odd numbered saccharide units of about 5 to 23 saccharide units. knit, and the even-numbered oligosaccharide of the present invention is exposed to ozone under acidic conditions. Oligosaccharides prepared by removing terminal non-reducing sugars by treatment with Contains a series of complements of tucarides.

The following example describes the synthesis of oligosaccharides and their anticomplement and anticoagulant activity. exemplify gender. The examples are illustrative and not restrictive.

Implementation blaze Heparin-oligosaccharides were added to heparinase (0.25 units of purified enzyme, 50 units/■; or commercially available enzyme (0.23 units/■)] Ta. Hevarinase was purified by 37.5d of 250mM sodium acetate, 2.5mM acetic acid. 300μ of heparin (porcine mucosal heparin or horse) in calcium solution (pH 7.0) commercially available products such as pulmonary heparin). This mixture was heated at 30°C for 40 hours. Incubated for a while. Aliquots are removed periodically and the sample is ．． The absorbance was measured at 232 nm after diluting with 0.03N hydrochloric acid at 100%. After % completion, stop the reaction by heating the reaction mixture to 100″C for one field. I let it happen. The product was frozen at -70°C, lyophilized, and redissolved in distilled water. The capacity was set to 4d. Next, the redissolved sample was placed on Sephadex G-10. desalted, collected, lyophilized, and redissolved in 6 ml of distilled water to a final concentration of 50 ■/d.

Partial enzymatic depolymerization of heparin was controlled and reconstituted at 30% reaction completion. It shows a certain heparin-oligosaccharide distribution, and the mixture is about 10 to Maximum number of heparin-oligosancharides with a degree of polymerization of approximately 16 saccharide units and concentrations. This mixture also has a weight of 2 to 10 saccharide units. Heparin-oligosancalide and 16 to 24 saccharide units with a degree of Heparin-oligosaccharides with a degree of polymerization of It contained a sufficient amount. Heparinol with a degree of polymerization of 2 to 10 saccharide units If rigosatucharide is required, it is best to stop the reaction at 50-75% completion. preferable. Heparin-oligosanka with a degree of polymerization of 16 to 24 saccharide units If a ride is required, it is preferable to stop the reaction at 10-20% completion.

Real] l quick entry This example illustrates the fractionation of heparin-oligosaccharides. Hebarinoo υ Gosa, callide mixture (20mg) was added to 0.2M sodium chloride (pH 3,5 ) to a strong anion exchange HPLC semi-preparative column pre-equilibrated with 1.5 d/min. It was applied at a flow rate of using sodium chloride (pH 3,5) at 1.5 mfl for 1 minute. A linear salt gradient was started immediately. Both fractions from each peak were collected, lyophilized, Redissolve in 4 ml of distilled water and 3I on a 3 cm x 45 cm column. By Sephadex G-10 gel filtration chromatography at a flow rate of n17 min. (samples with degree of polymerization less than 6), or permeability to 3 x 1000 volumes of deionized water. Desalting was carried out by analysis (samples with degree of polymerization greater than 6). Lyophilize the sample and It was then stored at -70°C.

have the desired degree of polymerization and contain a single major component as a result of charge-based fractionation A purified heparin-oligosaccharide sample was obtained. It is shown in Figure 1. Therefore, the elution order from the anion exchange-HPLC column was dependent on the degree of polymerization (i.e. First, disaccharide (2), followed by tetrasaccharide, followed by hek. Sasatsuka Ride, etc.). Within each size group, the elution order depends on the degree of sulfation. dependent (i.e., tetrasaccharide 4A with 5 sulfates and Royal, followed by a tetrasancharide with six sulfates). child The fractionation method uses purified hebarin with a polymerization degree of 2 to 24 saccharide units. Suitable for preparing oligosaccharides.

Mishin ■ Kimoto This example illustrates the quantification and analysis of heparin-oligosaccharides. Freeze drying Dissolve the dried sample in distilled water to a volume of one precept and take an aliquot of each. The solution was taken out, added to 0.03N hydrochloric acid, and the absorbance was measured at 232 nm. next, The concentration of the sample was calculated based on its molecular weight. Molecular weight is determined directly from its chemical structure Calculate or gel permeation HPLCC3 Harath et al. See Near 3-83 (1985)] and 5,2 x 10”M-’cm. It was calculated from its degree of polymerization determined using the particle extinction coefficient.

More concentrated samples may be diluted further to ensure that all samples are approximately the same concentration (1 ■/Single). Quantification of stock solutions of 1 μ/d is performed by preparing them. Carbazole assay against standard curve prepared using heparin used for Uronic acid was measured by A. Compositional analysis is shown in Table 1.

Implementation five balls This example demonstrates the use of heparin and The activity of heparin-oligosaccharide is shown. Weiler et al., 2bl Moon Sil I Yu, 147:409-421.1978; Weiler.

Immuno harmacolo, 6:245-255, 1983; and 5 Harath et al., Tori” and Anal Residence ■, Uni 73-83. 1983. The method used was used. In summary, large, moderate and small amounts of 0 on the cell surface Inhibition was tested in experiments using EAC4b, 3b prepared to have 3 I tried it. 109 EAC4b, 100i per 2a cellular intermediate, 20μg and EAC4b, 3b”0.EAC4 using 5 μg of 03 input, respectively. b, 3b”, and EAC4b, 3b5 were prepared. Complement assay buffer (DGV B"a 100 p! only used for reagent blank, non-inhibition and 100% lysis. or heparin or heparin-oligosancalide Add solution containing complement assay buffer to the remaining tube. At time 0, I Suspension of XIO' EAC4b, 3b, excess amount of P and D (100ngD) , and the amount required to lyse the uninhibited tube with an average of one hemolytic event per cell. 100μ of complement assay buffer containing B! (About EAC4b, 3bl Go 0.03ng of glue, 0.15ng of B1 for EAC4b, 3b" For EAC4b and 3b5, 0.50 ng of B) was added to each tube.

The mixture was incubated at 30°C for 30 minutes with stirring. Next, 40mM Add 300 I of rat serum 20 diluted in EDTA to the tube and stir. Incubation was continued for 60 minutes at 37°C without stirring. Next, the salt solution ( 1.5mf) to each tube, except for the 100% tubes, where 1.5mf) was added instead of the salt solution. , 5, and water were added to perform dissolution. Finally, mix the contents of the tube and centrifuge. The absorbance of the supernatant was measured at 414 nm.

Next, % lysis, average number of hemocyte lysis sites per cell (Z), and % inhibition were calculated.

Data are presented as % inhibition or ED50 for heparin (50% inhibition ED50 for heparin-oligosaccharide It is expressed as divided by 100 times. Heparin and heparin-oligosatsuka Concentrations of ride are expressed as cod per lXlO' cell intermediate. compertase production The volumes of the reaction tubes varied between 0.2 and 0.3. Number of cell intermediates was kept at 1 x 107 per tube. Lysis seen in non-inhibited tubes (Z) is relative to the volume of buffer present in the tube during compertase production. It was relatively independent. Similarly, the amount of inhibition of compertase production was independent of the amount of buffer present in the tube during the formation.

The results of this experiment indicate that as C3b loading increases, heparin The inhibitory activity of the drug decreased. In this experiment, the average hemolytic cell lysis per cell was maintained. The amount of B input decreases as the concentration of C3b on cellular intermediates decreases to increased. This experiment demonstrated that EAC4b, 3b in the presence of low, intermediate and high concentrations of B. To determine the degree of inhibition of heparin that prevents effective compercuse assembly on It was determined whether only the B input played a role. present in cellular intermediates The amount of B to inhibit the ability of heparin or heparin-oligosaccharide to inhibit dissolution. It had no effect on the

The results of the experiment shown in Figure 2 are based on disaccharides and tetrasaccharides with the sequences defined in Table 1. Heparin using Ride Artificial, Tutu and Wangli, and 1q Hexasaccharide - Shows the regulation of complement activation by oligosaccharides. Oligosaccharides are complementary Linear extenuating response curve of 1 to 8 cod per cell intermediate, showing low activity for body activation. (Figure 2). Next, 6-1 with molecular weight in the range of 1892-5320 Regarding the activity of heparin-oligosaccharides with a degree of polymerization of 6 saccharide units, It was tested. The results were compared to the activity of native heparin on a weight basis. high molecular weight Heparin-oligosancalide (16B, molecular weight 5320) is a naturally occurring It was 54% of the activity of heparin and 130% of the heavy MTi level.

Heparin oligosancalide has as many as 24 sancalide units. Prepared according to the methods described herein. This oligosaccharide has very low It had an anticoagulant activity lower than that of native heparin (on a weight basis). (defined as less than %). Heparin-oligosancalides are described herein. were tested for their ability to control complement activation. 24 Satsuka Ride Unit The oligosaccharide with 100% was equivalent to heparin on a molecular basis.

This example also demonstrates the relationship between oligosancalide structure and complement regulatory activity. F Pure hexafluoride of defined composition ranging from xasatucalides to decasatucalides. Eleven parin-oligosancalides were tested (Figure 4), and these homogeneous heparin Phosphorus-oligosancalides have lower anticoagulant properties than their less purified counterparts. activity and substantially the same complement activation inhibitory activity.

Burning ceremony This example demonstrates the anticoagulant activity of each heparin and heparin oligosancalide fragment. vinegar. Anticoagulant activity is measured by aPTT (activated thromboplasmin time) (Linh Ardt et al., J. Biol, Chem, 2￥7 Near 310-13.1982 ) was determined. Antithrombin ■ - Mediating antifactors - Ha and antifactors Tar-Xa activity was purified as described in Linhardt et al. and Table 2. It was measured using plasma protein. As shown in Figure 5, five types of porcine heparin are It exhibited 90-107% activity of standard porcine heparin (Hl) on a quantitative basis. 2 types Bovine heparin has 106% and 124% of standard porcine heparin activity, and Molecular weight heparin has 100% of the activity of standard porcine heparin for complement activation was.

Activated partial thromboplastin time assay and antithrombin m-mediated Heparin-oligosaccharide and bioactivity using anti-factor 11a assay The anticoagulant activity of commercially available heparin gave similar results, and the anti-factor Ua assay It showed a much lower value (Table 2). determined by anti-factor-Ua assay The anticoagulant activity of heparin-oligosaccharides and commercially available heparin is inhibited by complement activation. Their ability to harm is plotted in Figure 5. porcine heparin, bovine heparin Heparin and low molecular weight heparin are grouped together and show only slight differences in activity. . On the other hand, heparin oligosaccharides show that the complement rate increases as the degree of polymerization increases. It showed a slight increase in anticoagulant activity accompanied by a significant increase in anticoagulant activity.

Nonreduction of heparin oligosaccharides with an even number of saccharide units A method for removing the Δυ static sancalide unit from the terminus is shown. This method works for odd numbers Generate complementary groups of heparin-oligosaccharides with saccharide units. Close. 0.6 to 24 saccharide units of heparin-oligosancalide. It was dissolved in 0.03N hydrochloric acid at 100 cod/d. Until the solution stops absorbing light at 232 nm. Then, ozone gas was bubbled through this liquid. Adjust the pH of this solution to pH 7.0 and the low molecular weight fragment of the ΔUAp saccharide unit was transferred to Sephadex 6. -15 column by gel permeation chromatography. odd number satsuka Recover heparin oligosancalide with ride unit and uronic acid Quantitate using

Although the invention has been described by way of illustration and examples for purposes of clarity and understanding, Certain changes and modifications may be made without departing from the spirit and scope of the invention. cormorant.

Pure! (No change in content) γ　几　T’itl＆　日　＼1J-N! 6. Subject of correction (1) Translation of the description and claims (2) Translation of drawings (3) Power of attorney 7. Contents of correction (1) Translation of the description and claims (no change in content) (2) Engraving of the translation of the drawing (no changes to the content) (3) As shown in the attached sheet 8. List of attached documents (1) Translation of the specification and claims (1 copy each) (2) Translation of the drawings (1 copy) (3) Power of attorney and its translation: one copy each international search report PCT/US 89103312 Car・°・～・P“＋＊elAssμ・・1・・ト・・−2−

Claims (10)

[Claims] 1. The following formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (wherein R is a metal cation or a nonmetal cation, X is SO3R, and and n is an integer from 5 to 11); Has heparin-like anti-complement activity; Has reduced anticoagulant activity compared to heparin; and the sodium salt is at least An oligosaccharide characterized in that it contains 14% sulfur. 2. The oligosaccharide according to claim 1, wherein R is selected from the group consisting of Na, K and Li. Calaride. 3. Oligosaccharide according to claim 1 or 2 and acceptable for parenteral administration A pharmaceutical composition comprising a pharmaceutical carrier. 4. Anticoagulants with heparin-like anticoagulant activity and reduced anticoagulant activity compared to heparin A method for producing ligosaccharides, in which heparin is partially processed using the enzyme heparinase. depolymerize to obtain a polysaccharide mixture; The polysaccharide mixture was fractionated and separated by an anion exchange column. Collect fractions; The separated polysaccharide fraction was treated with heparinase to give 2,4A, Disaccharides identified as 4B, 4C and 6C, tetrasaccharides and producing hexasaccharides; and By polymerizing the disaccharide, tetrasaccharide or hexasaccharide, 1 Form oligosaccharides with even degree of polymerization from 0 to 24 saccharide units a method that involves: 5. exposing the oligosaccharide with an even degree of polymerization to ozone in an acidic environment; Further, the terminal non-reducing sugars are removed to form odd-numbered oligosaccharides by 5. The method of claim 4, comprising: 6. The following formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (wherein R is H, or a metal cation or non-metal cation, and X is H or SO2R, X is H, COC1-6 alkyl or SO3R, and n is an integer from 1 to 11); has heparin-like anticomplement activity; and Odd oligosaccharide characterized by having reduced anticoagulant activity compared to heparin Calaride. 7. Claim 6, wherein R is selected from the group consisting of H, Na, K and Li. Odd-numbered oligosaccharides described in . 8. Odd oligosaccharide according to claim 6, wherein Y is COCH3. 9. 7. The odd oligosaccharide of claim 6, wherein n is an integer from about 4 to about 10. 10. Odd-numbered oligosaccharide according to any one of claims 6 to 9, and as a pharmaceutical A pharmaceutical composition comprising an acceptable carrier or diluent.